Understanding the complex regulation of beta-globin genes is critically important to design therapeutic approaches to beta-thalassemia and sickle cell disease. An erythroid-specific protein complex containing LDB1/GATA-1/TAL1/LMO2 (LDB1 complex) is directly involved in activation of globin gene expression through providing long-range interaction between the beta-globin LCR enhancer and globin gene promoters. Additionally, beta-globin gene expression is regulated by the G9a methyltransferase, which is responsible for establishing histone H3K9 dimethylation (H3K9me2). However, the mechanistic role of G9a and its functional interaction with the LDB1 complex in regulation of beta-globin gene expression remain unclear. Using ex vivo differentiation of primary CD34(+) adult human cells as a model system, we found that repression of G9a activity by the chemical compound UNC0638 during the most proliferative phase of CD34(+) cell differentiation caused a strong increase in gamma-globin gene expression as well as reduction of beta-globin gene expression without substantial change in alpha-globin gene expression. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus, gain of LDB1 complex occupancy at the gamma-globin promoters as well as de novo formation of LCR/gamma-globin contacts. These results support a model whereby G9a establishes H3K9me2 conditions preventing interaction of the gamma-globin gene with the LCR and, instead, facilitates LCR looping with beta-globin gene promoters and results in their strong activation during differentiation of adult erythroid progenitor cells. In this view, G9a inhibition represents a promising approach for treatment of beta-globin hemoglobinopathies by reactivating fetal gamma globin gene expression at the expense of the adult genes in adult erythroid cells. The results provide an example of reconfiguring chromatin loops by epigenetic modification to alter gene expression. Cell type specific regulatory elements, enhancers and promoters, control lineage specific transcriptomes. Our current understanding is that lineage-specific transcription factors and complexes that binds to these regulatory elements, can orchestrate their long range interactions, for example, by protein dimerization. In other cases, lineage-specific factors co-opt ubiquitous architectural looping proteins such as CTCF into cell type-specific enhancer long range interactions, but in these cases molecular details are unclear. We identified a region upstream of carbonic anhydrase 2 (Car2) that is strongly occupied by the looping protein LDB1 and determined it to be a Car2 enhancer by genome editing with CRISPR-Cas9. In erythroid cells, the enhancer contacts Car2 when it is highly transcribed and the Car2 promoter is occupied by CTCF, but not by LDB1. LDB1 and CTCF interact directly to mediate enhancer looping while knockdown of either protein abrogates the enhancer loop and reduces Car2 transcription. Moreover, we identify a subset of CTCF-occupied genes that interact with LDB1-bound known or putative enhancers in erythroid cells. CRISPR/Cas9 deletion of select enhancers compromises gene transcription, validating enhancer function and generalizing the importance of LDB1-CTCF interaction in enhancer looping and establishment of the erythroid transcriptome. The principles underlying the architectural landscape of chromosomes in living cells remain largely unknown despite its potential to play a role in mammalian gene regulation. We found that the 3-dimensional conformation of a 1 Mbp region of human chromosome 11 containing the beta-globin locus functionally contributes to globin gene expression according to a polymer model of CTCF-dependent chromosome folding. To test this function in vivo, we are using CRISPR/Cas9 technology to delete CTCF sites such as HS5 and 3'HS1 (flanking the beta-globin locus) and CTCF sites between or in the boundaries of CTCF/cohesin-mediated chromatin domains in erythroid K562 and non-erythroid 293T cells. PCR amplification and sequencing were used to validate mono- and bi-allelic deletions. Western blots showed that CTCF protein level remained the same in CTCF binding site deleted cells. ChIP indicated that CTCF binding site deletion resulted in loss of CTCF binding at these sites. Interestingly, deletion of one CTCF motif was capable of reducing CTCF occupancy at other CTCF sites, possibly by affecting long range interactions as revealed by 3C. RT-PCR revealed changes in gamma globin gene expression associated with CTCF site deletion. To understand how CTCF binding site deletion alters beta-globin gene expression, RNA-seq analysis is being carried out in these two cell lines. Further study using Chromosome Conformation Capture Hi-C will bring us a closer look at the function of CTCF in cell type specific chromosome architecture organization.